Photoprotein derived from okinawan squid and gene encoding the photoprotein

ABSTRACT

By using a bioluminescence, a method for detection of a monovalent cation is provided and the method is a high sensitive and simple method. According to the present invention, an amino acid sequence of symplectin and a base sequence encoding the protein are provided. Symplectin is a photoprotein derived from  Symplectoteuthis oualaniensis  (okinawan squid). By using the photoprotein of the present invention, a monovalant cation can be detected by luminescence in the presence of a chromophore.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to symplectin, which is a photoproteinderived from okinawan squid (Symplectoteuthis oualaniensis: Tobi-Ika).Moreover, this invention relates to an amino acid sequence of symplectinand a base sequence of gene encoding the protein. Furthermore, thepresent invention relates to a method for detection of a monovalentcation by monitoring luminescence, generated using said photoprotein inthe presence of a chromophore.

[0003] 2. Prior Art

[0004] Bioluminescence has been applied for various purpose, such asmonitoring of concentration of a metal ion in a living cell, becausesome complementary factor is requisite for generation of luminescence. Achromophore is oxidized and then a high energy state intermediate(luminescent intermediate) is formed. It collapses to form a basal stateand emission of luminescence occurs accompanied with such alteration ofenergy state. In a photogenic organism, emission of luminescent occursefficiently using an enzyme.

[0005] The most classical bioluminescence is observed in firefly, whichis well-known as luciferin-luciferase reaction. In luciferin-luciferasereaction, luciferin is converted to oxyluciferin via enzymatic reactionof luciferase in the presence of ATP and magnesium ion. It is theessential phenomenon involved in the photo-reaction. As the reactionmediated by firefly luciferase enables detection of luminescence withextremely high sensitivity, it is an important tool for investigation inthe field of biochemistry. The gene encoding firefly luciferase has beenalready cloned. Then, heat-stable type luciferase is produced by geneticengineering technique using E. coli and it is commercially available.

[0006] For another example, aequorin, a blue fluorescent protein ofjellyfish (Aequorea victoria) capable of emitting blue colorluminescence, has been also known. Aequorin has a relatively lowmolecular mass of 21 kDa. Aequorin uptakes a chromophore such ascoelenterazine and oxygen, then it is converted to an exited state (highenergy state) using calcium ion as a trigger, thereby emits blue colorluminescence. The color of jellyfish luminescence is actually blue colorand not green color. However, green fluorescent protein (GFP) is assumedto receive energy from aequorin, thereby emitting green colorluminescence.

[0007] The above luminescent system of jellyfish has been applied in thefield of cell physiology and biochemistry. Emission of luminescencecaused by aequorin is triggered by calcium ion. Then aequorin has beenutilized to detect alteration of calcium ion concentration, such as foranalysis on alteration of cytoplasmic calcium ion concentration causedby agonistic stimulation of intact cells, for example, by varioushormones, agonists such as neurotransmitters or growth factors.Therefore, aequorin has attracted attention as a calcium sensor or agene reporter, in the field of clinical biochemistry and cellphysiology.

[0008] The above-mentioned method, utilizing luciferin or aequorin, isan excellent method for its high sensitivity. However, the ions whichserves as the trigger of luminescence are magnesium ion and calcium ionfor luciferin and aequorin, respectively. These are both divalentcations. Thus, there has been no means which enables detection of amonovalent cation using a photoprotein. The method utilizing aphotoprotein is advantageous, because its sensitivity is extremely highand it is free from danger like experiments using a radioisotope.Therefore, a photoprotein which can detect a monovalent cation has beendesired.

SUMMARY OF THE INVENTION

[0009] Thus, the inventors have noticed bioluminescent system ofokinawan squid (Symplectoteuthis oualaniensis: Tobi-ika) from Okinawa,Japan. The luminescence of the squid was trigged by monovalent cationssuch as potassium, sodium or etc. and the inventors investigated on theluminescent system. In the luminescent system of okinawan squid, amonovalent cation acts as a trigger. Thus, the luminescent proteinderived from okinawan squid is considered to be an excellent tool fordetection of a monovalent cation.

[0010] One aspect of this invention of a protein derived fromSymplectoteuthis oualaniensis (okinawan squid) having the followingcharacteristics:

[0011] (1) emitting luminescence at wavelength of 470 nm by binding todehydrocoeleneterazine or a dehydrocoeleneterazine derivative in thepresence of potassium ion or sodium ion:

[0012] (2) having a molecular mass of approximately 60 kDa analyzed bySDS-polyacrylamide gel electrophoresis:

[0013] (3) dissolving into a solvent containing KCl at a concentrationof 0.6M or higher while maintaining ability to emit luminescence: and

[0014] (4) decomposing by trypsin digestion to produce fragments havingmolecular masses of 40 kDa and 15 kDa.

[0015] According to the present invention, said dehydrocoeleneterazinderivative can be a compound selected from the group consisting ofcompounds represented by the following formula (1), (2) or (3):

[0016] wherein X is a halogen atom or methoxy group, a compoundrepresented by the following formula:

[0017] wherein X represents a halogen atom, methoxy group or hydroxylgroup, and Y and Z each represents hydrogen atom, a halogen atom,methoxy group or hydroxyl group, with the proviso that a case where bothof Y and Z are hydrogen atoms is excluded, and

[0018] a compound represented by the following formula:

[0019] wherein X and A each represents a halogen atom, methoxy group orhydroxyl group, and Y, Z, B and C each represents hydrogen atom, ahalogen atom, methoxy group or hydroxyl group, with the proviso thatcases where Y and Z are substituents selected from an atom or groupsdescribed above with both of B and C being hydrogen atoms and the casewhere all of Y, Z, B and C are hydrogen atoms are excluded.

[0020] Further aspect of this invention is a protein derived fromSymplectoteuthis oualaniensis (okinawan squid) consisting of an aminoacid sequence of following (a) or (b):

[0021] (a) an amino acid sequence represented by amino acid numbers 1 to501 shown in SEQ:ID NO:1 in a sequence list, or (b) an amino acidsequence in which a part of said amino acid sequence (a) is deleted oranother amino acid sequence is added to said amino acid sequence (a) ora part of amino acid sequence (a) is substituted with another amino acidsequence, the amino acid sequence (b) having characteristics that bindswith dehydrocoeleneterazine by covalent bonding in the presence ofpotassium ion or sodium ion thereby emitting luminescence. Thefunctional 40 kDa fragment of this protein, represented by amino acidnumbers 1 to 370 shown in SEQ:ID NO:2 in a sequence list, is also withinthe range of this invention.

[0022] Moreover, further aspect of this invention is a method fordetection of a monovalent cation, the method comprising formation of aconjugate adduct comprising the protein described above and anchromophore, thereby emitting luminescence from said conjugate adduct inthe presence of a monovalent cation.

[0023] According to the present invention, said chromophore can be acompound selected from the group consisting of compounds represented bythe following formula (4), (5), (6) or (7):

[0024] compound represented by the following formula:

[0025] wherein X is a halogen atom or methoxy group, a compoundrepresented by the following formula:

[0026] wherein X represents a halogen atom, methoxy group or hydroxylgroup, and Y and Z each represents hydrogen atom, a halogen atom,methoxy group or hydroxyl group, with the proviso that a case where bothof Y and Z are hydrogen atoms is excluded, and

[0027] a compound represented by the following formula:

[0028] wherein X and A each represents a halogen atom, methoxy group orhydroxyl group, and Y, Z, B and C each represents hydrogen atom, ahalogen atom, methoxy group or hydroxyl group, with the proviso thatcases where Y and Z are substituents selected from an atom or groupsdescribed above with both of B and C being hydrogen atoms and the casewhere all of Y, Z, B and C are hydrogen atoms are excluded.

[0029] Moreover, further aspect of this invention is a gene derived fromSymplectoteuthis oualaniensis (okinawan squid) consisting of a basesequence of following (e), (f) or (g):

[0030] (e) a base sequence represented by base numbers 1 to 1646 shownin SEQ:ID NO:3 in a sequence list,

[0031] (f) a base sequence in which a part of said base sequence (e) isdeleted or another base sequence is added to said base sequence (e) or apart of base sequence (e) is substituted with another base sequence, thebase sequence (f) encoding a protein having characteristics that bindswith dehydrocoeleneterazine by covalent bonding in the presence ofpotassium ion or sodium ion thereby emitting luminescence, or

[0032] (g) a base sequence that hybridizes with the base sequence (e)under stringent conditions.

[0033] Moreover, further aspect of this invention is a method fordetection of a monovalent cation, the method comprising introduction ofthe gene described above into a cell, expression of protein encoded bysaid gene in the cell, formation of a conjugate adduct of the proteinand a chromophore, thereby emitting luminescence from said conjugateadduct in the presence of a monovalent cation.

[0034] According to the present invention, said chromophore can be acompound selected from the group consisting of compounds represented bythe following formula (8), (9), (10) or (11):

[0035] a compound represented by the following formula:

[0036] wherein X is a halogen atom or methoxy group, a compoundrepresented by the following formula:

[0037] wherein X represents a halogen atom, methoxy group or hydroxylgroup, and Y and Z each represents hydrogen atom, a halogen atom,methoxy group or hydroxyl group, with the proviso that a case where bothof Y and Z are hydrogen atoms is excluded, and

[0038] a compound represented by the following formula:

[0039] wherein X and A each represents a halogen atom, methoxy group orhydroxyl group, and Y, Z, B and C each represents hydrogen atom, ahalogen atom, methoxy group or hydroxyl group, with the proviso thatcases where Y and Z are substituents selected from an atom or groupsdescribed above with both of B and C being hydrogen atoms and the casewhere all of Y, Z, B and C are hydrogen atoms are excluded.

[0040] Moreover, further aspect of this invention is a transformedEscherichia coli wherein the gene described above is introduced.

[0041] Moreover, further aspect of this invention is a method to producea recombinant protein, the method comprising introduction of the genedescribed above succeeded by expression of protein encoded by said genein Escherichia coli, said recombinant protein binds withdehydrocoeleneterazine by covalent bonding in the presence of potassiumion or sodium ion to form a conjugate adduct, thereby emittingluminescence from said conjugate adduct.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1 is a schematic view showing the assumed mechanism ofemission of luminescence observed in okinawan squid.

[0043]FIG. 2 is a photograph showing the result of SDS-PAGE analysis ofthe present photoprotein, with and without subjection to trypsindigestion.

[0044]FIG. 3 is a figure showing strategy of PCR cloning, utilized toobtain cDNA of the present photoprotein.

[0045]FIG. 4 is a figure showing schematic view of structure of thepresent photoprotein and hydropathy profile.

[0046]FIG. 5 is a figure showing primers and template utilized for PCRamplification.

[0047]FIG. 6 is a figure showing strategy for PCR amplification of thecDNA of the present photoprotein.

[0048]FIG. 7 is a photograph showing the result of Western blot analysisof the obtained recombinant protein.

DETAILED DESCRIPTION OF THE INVENTION

[0049] Okinawan squid has a large photogenic organ on its body. Here, aphotoprotein symplectin is involved in the luminescence emitting system.Dehydrocoeleneterazine is bounded to symplectin protein by covalentbonding and an oxygen molecule and a monovalent cation (such aspotassium, sodium, etc.) are required for emission of luminescence. Thatis, dehydrocoeleneterazine (chromophore) is incorporated into theprotein to form a conjugate adduct by covalent bonding. Then thechromophore is converted to its reduced form in the presence of amonovalent cation, which result in emission of luminescence. It isassumed that, conformation of the photo-protein is altered in thepresence of a monovalent cation, whereby a chemical reaction accompaniedwith emission of luminescence occurs. This is the reason why amonovalent cation serves as a trigger of luminescence in thephoto-system of okinawan squid. The assumed luminescent mechanism ofokinawan squid, clarified using a model system of glutathione, is shownin FIG. 1.

[0050] In the luminescent system of okinawan squid,dehydrocoeleneterazine can be used as a chromophore anddehydrocoeleneterazine binds to symplectin, the photoprotein of thisinvention.

[0051] Incidentally, the inventors have utilized a system using crudeapo-protein without a chromophore and added the above-mentionedchromophore or its derivative to the system, thereby reconstituted thephotoprotein. As the result of such experiment, emission of luminescencewas confirmed.

[0052] The photoprotein of okinawan squid has a low solubility and thereis no conventional method for research of the protein. When a surfactantto solubilize the membrane protein is used, luminescent activity of theokinawan squid photoprotein would be disrupted. By addition of KCl (acaotropic salt) to a buffer, the protein can be solubilized withoutdisrupting its activity. The effects of various kinds of salts on theobjective protein are examined in the terms of extraction efficiency andluminescent activity. As the result, it was found that KCl was the mostsuitable. Also, different from the case of aequorin, luminescence ofsymplectin was not triggered by a divalent cation. Enzymaticcharacteristic of okinawan squid was investigated. As a result, it wasfound that the optimum pH was 8 and the optimum temperature was about20° C. (Isobe M. et al., Pure and Appl. Chem., (1998) Vol.70,2085-2092).

[0053] As described in the following Examples, the present inventorshave extracted the photoprotein derived from okinawan squid in a buffercontaining KCl. Moreover, the inventors have determined the basesequence by PCR cloning and determined the amino acid sequence. Thestructure of symplectin of the present invention, which is aphotoprotein derived from okinawan squid, can be specified by the aminoacid sequence described in Sequence ID. NO:1 of the sequence List. Theabove-mentioned chromophore is covalently bonded to the protein shown inthe amino acid sequence, then emission of luminescence occurs in thepresence of a monovalent cation as a trigger. The protein specified bysaid amino acid sequence shows a molecular mass of about 60 kDadetermined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0054] The polypeptide consisting of an amino acid sequence in which apart of said polypeptide specified by amino acid sequence shown in SEQID NO: 1 is deleted, substituted or added with another amino acidsequence means a polypeptide in which 20 or less, preferably ten orless, and more preferably five or less amino acids of the sequence isdeleted, substituted or added to the amino acid sequence shown in SEQ IDNO: 1 in a sequence list. Moreover, such polypeptide exhibits homology70% or more, preferably 80% or more and still preferably 90% or morewith the amino acid sequence shown in SEQ ID NO: 1 in a sequence list.Such polypeptide is also within the range of this invention so far as itexhibits the characteristic as the photoprotein symplectin, that bindswith a chromophore by covalently bonding in the presence of a monovalentcation thereby emitting luminescence.

[0055] Also, as shown in the following Examples, treatment of saidprotein with trypsin results in decomposition into two fragments of 40kDa and 15 kDa. Here, the 40 kDa fragment, corresponding to C-terminalportion of the original protein, is involved in the photogenic functionof the protein. Amino acid sequences of the above-mentioned 40 kDaobtained by the trypsin digestion is shown in SEQ ID NO: 2 in a sequencelist. The polypeptide having the amino acid sequence shown in SequenceID. NO: 2 in a sequence List is also within the scope of the presentinvention.

[0056] The polypeptide consisting of an amino acid sequence in which apart of said polypeptide specified by the amino acid sequence shown inSEQ ID NO: 2 is deleted, substituted or added with another amino acidsequence means a polypeptide in which 20 or less, preferably ten orless, and more preferably five or less amino acids of the sequence isdeleted, substituted or added to the amino acid sequence shown in SEQ IDNO: 2 in a sequence list. Such polypeptide is also within the range ofthis invention so far as it exhibits characteristic as the photoproteinsymplectin, that binds with a chromophore by covalent bonding in thepresence of a monovalent cation thereby emitting luminescence.

[0057] Moreover, cDNA of the gene encoding symplectin protein isspecified by the base sequence shown in SEQ ID NO: 3 is a sequence list.The base sequence described in SEQ ID. NO:3 in a sequence list is a basesequence corresponding to its full length cDNA. The sequence includesopen reading frame that encodes 489 amino acids residues, whichcorresponds to the region from the thirteenth residue phenylalanine toserine residue of the C-terminal shown in SEQ ID. NO: 1 in a sequencelist.

[0058] With regard to many amino acids, one amino acid is encoded byplural base sequences of DNA. Therefore, plural genes, other than nativegene of this invention, might encode amino acid sequence of symplectinidentified by the present invention. The gene of this invention is notto be limited to only native gene and intended to include many othersequences encoding the photoprotein identified by the present invention.

[0059] According to technique of gene recombination, artificialmodification can be achieved at a specific site of basic DNA, withoutalteration improvement of basic characteristic of said DNA. Concerning agene having native sequence provided according to this invention ormodified sequence different from said native sequence, it is alsopossible to perform artificial modification such as insertion, deletionor substitution to obtain gene of equivalent or improved characteristiccompared with said native gene. Moreover, a gene with such mutation isalso included in the range of this invention. That is, the gene,consisting of a base sequence hybridizes with said base sequence shownin SEQ ID NO: 3 in a sequence list under stringent condition, means agene in which 20 or less, preferably ten or less, and more preferablyfive or less bases of the sequence is deleted, substituted or added tothe base sequence shown in SEQ ID NO: 3 in a sequence list. Moreover,such gene exhibits homology 70% or more, preferably 80% or more andstill preferably 90% or more with the base sequence shown in SEQ ID NO:3 in a sequence list. In addition, such gene hybridizes with the basesequence shown in the SEQ ID NO: 3 in a sequence list under stringentcondition. Such gene is also within the range of this invention so faras it encodes a polypeptide having characteristic as the photoproteinsymplectin, that binds with a chromophore by covalent bonding in thepresence of a monovalent cation thereby emitting luminescence.

[0060] The photoprotein of the present invention is particularly usefulfor detection of a monovalent cation with high sensitivity.Bioluminescence can be detected with extremely high sensitivity ofapproximately 10⁻¹⁶ mol. Therefore, it is possible to detect amonovalent cation rapidly easily and simply, with sensitivity equivalentto or higher than that of a radioisotope. Thus, the photoprotein of thepresent invention has excellent characteristics as a sensor availablefor detection of a monovalent cation, particularly potassium ion orsodium ion.

[0061] In concrete, the photoprotein of the present invention or thegene encoding the photoprotein can be utilized for a clinical reagentfor analysis in the field of medical treatment or for a research reagentfor biochemical research. In the field of clinical analysis, for thepurpose of in vitro (in a test tube) detection of a monovalent cation,the photoprotein of the present invention is particularly useful. Notonly for the above purpose, the photoprotein of the present inventioncan be utilized for detection of infinitesimal alteration of monovalentcation concentration in a cell. Aequorin, a photoprotein of theabove-mentioned jellyfish, has been used as an indicator for detectionof calcium ion concentration in a cell, as a conventional technique inthis technical art. It is assumed that the photoprotein of the presentinvention can be utilized as an indicator for detection of a monovalentcation, in the manner similar to aequorin. In regard to aequorin, atechnique applicable for detection of alteration of calcium ionconcentration in an intact cell has been established, using a system inwhich a gene encoding aequorin is expressed in a cell. Concerning thegene and the polypeptide of the present invention, the base sequence andthe amino acid sequence have been determined by the present invention.Therefore, they would be utilized in the same manner as aequorin.Particularly, for the investigation on the function of a nerve cell, itis very important to detect infinitesimal alteration of potassium ionconcentration. For example, alteration of an infinitesimal amount ofmonovalent cation in a cell can be measured by introduction of thephotoprotein of the present invention, using a technique such as microinjection or etc. Also, the same effects can be obtained by introductionof the gene encoding the photoprotein of the present invention,succeeded by expression of the gene. In these cases, a chromophore canbe provided externally. In a plant, particularly, the chromophore can beabsorbed from its root.

[0062] Incidentally, the present inventors have established a method fordetection of phosphatase activity with high sensitivity, usingluciferin-luciferase system of firefly. Luciferin phosphate is acompound wherein phosphate group is bonded to luciferin, which is achromophore. When luciferin phosphate was treated by phosphatase, achromophore luciferin was formed, enzymatic reaction by luciferase wasperformed using the formed luciferin as a substrate, then emission ofluminescence can be detected. Meanwhile, luciferin phosphate does notserve as a substrate of luciferase and generation of luminescence doesnot occur. That is, in the absence of luciferin formed by the enzymaticreaction by phosphatase, luminescence can not be detected. Therefore,the luminescent intensity correlates with the phosphatase activity. Thatis, enzymatic activity of phosphatase can be displaced to intensity ofbioluminescence.

[0063] The okinawan squid photoprotein of the present invention can bealso utilized for measurement of enzymatic activity of an enzymeaccording to the same principle. Explaining measurement of phosphataseactivity using an example, the method comprises formation of achromophore derivative, in which phosphate group is bonded to thechromophore such as dehydrocoelenterazine to form a substrate ofphosphatase, succeeded by performance of enzymatic reaction using thederivative as a phoaphatase substrate. The chromophore formed by theenzymatic reaction is covalently bonded to okinawan squid photoproteinand emits luminescence in the presence of a monovalent cation.Therefore, enzymatic reaction can be measured by detection ofluminescence. Measurement according to the same principle can be appliedto various kinds of enzymes other than phosphatase, so far as thechromophore can be converted to substrate of the objective enzyme.

[0064] In the following Examples, the present invention is explained inmore detail, but the present invention is not to be limited by the abovedescriptions nor the following examples. It should be noted that anyconventional modification known in this technical field is included inthe range of this invention.

EXAMPLES

[0065] From photogenic organ of okinawan squid, collected at the seaarea of Okinawa Prefecture Japan, the photoprotein having a molecularmass of 60 kDa was separated and purified as a luminescent ability as anindex. This photoprotein was soluble in a solution containing KCl at aconcentration of more than 0.6M. At first, homogenate of the photogenicorgan containing symplectin was washed by KCl solution (pH 6.0) of 0.4M.Then, it was extracted with a KCl solution of 0.6M, and a portionthereof was added to a buffer having pH of 8.0 to confirm theluminescent ability. Because of high salt concentration, means ofchromatography applicable for purification of the extract was limited.

[0066] When the purified protein was hydrolyzed by trypsin, two productshaving molecular masses of 40 kDa and 15 kDa were produced from theoriginal protein of 60 kDa (FIG. 2). In FIG. 2, lane 1 shows the resultoriginal protein before trypsin digestion, lane 2 shows that of trypsindigested crude protein, lane 3 shows that of trypsin digested purifiedprotein, and lane 4 shows that of molecular weight marker, respectively.Fragments of 40 kDa and 15 kDa were recognized after trypsin digestion,particularly in lane 3 corresponding to the purified protein. When theability to emit luminescence was investigated, both of 60 kDa protein ofand 40 kDa proteins exhibited luminescence. On the other hand, the 15kDa protein did not give luminescence. However, the amino acid sequenceof the 15 kDa protein was the same as the N-terminal amino acid sequenceof the 60 kDa protein. Then it was revealed that the 15 kDa proteincorresponded to the N-terminal sequence of symplectin. Incidentally, theactive region responsible for the photogenic function would exist at theC-terminal portion corresponding to the 40 kDa protein.

[0067] The protein thus purified was hydrolyzed by enzyme to producepeptide fragments, then partial amino acid sequence of these fragmentswere determined by analysis using nano-LC-Q-TOF-MS/MS. Chromatographyand mass-spectrum analysis (MS/MS spectrum) were carried out in suchmanner and partial amino acid sequences of several peptide fragmentswere determined. As the result, the N-terminal amino acid sequence wasfound to be YVRPVSSWK. Based on the partial amino acid sequences of thepeptide fragments thus determined, oligonucleotide primers weresynthesized to utilize for amplification of cDNA by PCR.

[0068] Next, from the photogenic organ of okinawan squid, RNA waspurified and cDNA pool was prepared by reaction utilizing reversetranscriptase. Using cDNA thus obtained as a template, polymerase chainreaction (PCR) was performed using the above-mentioned syntheticoligonucleotide primers and cDNA fragment of the photoprotein wasamplified. Sequences of the primers used for following PCR cloning wereshown below. 60(3)S: 5′-CGG GAT CCT TYG ARC AYC ARG TNA THC C-3′ (SEQ IDNO: 4) 40AS: 5′-GCC TCG AGT CRT TRT TRA ANG CNA CRT T-3′ (SEQ ID NO: 5)BLP-SP1: 5′-ATG GAC CTG ATG GCA GAG AA-3′ (SEQ ID NO: 6) MC-AS1: 5′-TGRTAD ATN GGN GGN ARG TTN AGG CA-3′ (SEQ ID NO: 7) FR29 AS: 5′-GTY TGN ACRTCR TCR TCY TC-3′ (SEQ ID NO: 8) photo-SP: 5′-GAGACCAGTACCTATGGTAGCGG-3′(SEQ ID NO: 9) Adapter primer: 3′-RACE cDNA synthetic primer

[0069] (SMART™cDNA Amplification kit: available from Clontech)

[0070] First, amplification by PCR was performed using 60(3)S and 40AS,primers designed based on the partial amino acid sequence. Based on thesequence, BLP-SP1, a primer specific for the gene, was synthesized.Moreover, based on the above-mentioned partial amino acid sequence twokinds of primers (MC-AS1 and FR29 AS) were synthesized. They werefurther elongated to the 3′ region. Finally, using the another primerspecific for the gene, 3′-RACE (rapid amplification of cDNA ends) wasperformed using SMART™cDNA amplification kit (available from Clontech)and the full length DNA was cloned. The strategy of the PCR cloningadopted for this experiment was shown in FIG. 3.

[0071] As a result, cDNA shown in Sequence ID. NO:3 in a sequence listwas obtained and it was comprised of 1646 bases. This cDNA contained abase sequence encoding a polypeptide comprising 489 amino acid residuesas the constructive gene, and the base sequence corresponds to the aminoacid sequence from the 13^(th) phenylalanine to serine at theC-terminal. The sequence comprising the twelve N-terminal amino acidsdetermined by the nano-LC-Q-TOF-MS/MS was added to the determinedsequence. As the result, full length amino acid sequence of okinawansquid photoprotein was determined and it was composed of 501 aminoacids. The amino acid sequence of symplectin thus obtained is shown inSequence ID. NO:1 in a sequence list. The calculated molecular mass ofthe polypeptide was 57,463 and the molecular mass was slightly smallerthan 60 kDa, which was evaluated by SDS-PAGE. Moreover, in the aminoacid sequence comprising the above-mentioned 501 amino acids, thesequence corresponding to the 40 kDa fragment is shown in Sequence ID.NO:2 in a sequence List. As described above, the 40 kDa fragmentobtained by trypsin digestion exhibited photogenic activity.

[0072] In the sequence of symplectin, a photoprotein of okinawan squid,there are two putative N-glycosylation sites. This notion indicates thepossibility that, this photoprotein might be a glycoprotein. The resultsof amino acid content analysis and hydropathy profile showed that thisprotein does not exhibit high degree of hydrophobic property, though thesolubility of this protein into an aqueous solution is low. A schematicdrawing of structure of symplectin is shown in the upper portion of FIG.4 and the result of hydrophacy profile is shown in the lower portion ofFIG. 4. The amino acid sequence of okinawan squid protein did notexhibit homology with the known photoproteins at all. The results showedthat this photoprotein derived from the squid was a novel photoprotein,as suggested from monovalent cation dependency for emission ofluminescence.

[0073] From homology research of the 60 kDa photoprotein using BLASTprogram, it was revealed that two proteins exhibited homology with thisphoto-protein. One of which is biotinidase (EC 3.5.1.12), a solubleenzyme which hydrolyzes biocytin to form biotin and lysine. Another oneis vanine, a GPI-anchord type protein. The membrane protein is expressedin perivascular thymic stromal cells, and suggested to be a homingreceptor for hematopoietic precursor cells to migrate into thymus. Onlylimited homology was recognized between the squid photoprotein and theseprotein derived from mammals. However, eleven cystein residues werepreserved in these three proteins.

[0074] The cDNAs encoding symplectin protein of this invention and 40kDa fragment thereof were amplified by PCR using following sense primers(PhPETS1, PhPETS2) and a common antisense primer(PhPETA1). As well,poly(+)RNA of the photogenic organ was utilized as a template for PCRamplification. As shown in FIG. 5, restriction sites (EcoRI site andSalI site) were inserted into these primers. Moreover, as shown in FIG6. cDNAs encoding full length symplectin protein of 60 kDa or its 40 kDafragment were amplified. PhPETS1: 5′-TTGAATTCCCAAAAACAGATATGGAGAC-3′(SEQ ID NO: 10) PhPETS2: 5′-TTGAATTCCTATTTTGGGAAGAAGGTTG-3′ (SEQ ID NO:11) PhPETA1: 5′-AAGTCGACTTAGGAGGCGGCGTAAACATAAG-3′ (SEQ ID NO: 12)

[0075] The PCR products were separated on an agarose gel succeeded bydigestion with EcoRI and SalI. Then it was ligated to EcoRI and SalIsite of pET-32a(+) to construct a vector. The vector was designatedpET-60 and it was utilized for expression of symplectin protein. The E.coli strain BL21(DE3)pLysS, transformed by pET-60, was grown in Luriabroth (LB) at 37° C. Expression of the protein was induced by additionof isopropyl-1-thio-b-D-galactopyranoside (IPTG). The cells wereharvested by centrifugation and lysed by sonication in a buffer. Thehomogenate was centrifuged, and the supernatant was subjected toaffinity chromatography using His-Bind Resin. The fusion protein withthioredoxin was purified by a step-wise manner elution with variedimidazole concentrations. The recombinant symplectin protein wasobtained as a fusion protein with thioredoxin, according to themanipulation described above.

[0076] As the result of SDS-PAGE of the cell lysate, symplectin and its40 kDa fragment were detected. Furthermore, Western blot analysis wasperformed and these bands were stained by anti-symplectin antibody. Itindicates that the protein according to this invention is expressed as arecombinant protein. The result of Western blot analysis is shown inFIG. 7. In FIG. 7, anti-symplectin antibody was used as the 1^(st)antibody and HRP-conjugated anti-mouse IgG was used as the 2^(nd)antibody. Moreover,lane 1 is recombinant protein of 40 kDa fragment,lane 2 is recombinant protein of full length 60 kDa protein and lane 3is crude extract. As indicated by arrows in FIG. 7, proteins exhibitingreactivity with the antibody was detected at the positions correspondingto 75 kDa and 55 kDa.

[0077] The recombinant symplectin protein was subjected tobioluminescence assay. The recombinant symplectin protein was suspendedin a buffer, mixed with chromophore dehydrocoeleneterazine, thenprotein-chromophore complex was formed by incubation at 37° C. for 30min. The alkaline buffer (pH9.8) was added to increase pH of theprotein-chromophore complex solution and the bioluminescence reactionwas initiated. The generated luminescence was monitored bylumiphotometer and obvious luminescence was detected as the result. Theability of recombinant symplectin protein to emit luminescence indicatesthat cDNA of symplectin protein actually encodes photoprotein derivedfrom okinawan squid. According to the present invention, an amino acidsequence of symplectin, a photoprotein derived from okinawan squid(Symplectoteuthis oualaniensis: Tobi-Ika) and a base sequence of thegene encoding said protein were provided. The photoprotein of thepresent invention enables detection of a monovalane cation by monitoringchemiluminescence in the presence of a chromophore.

1 12 1 501 PRT Symplectoteuthis oualaniensis 1 Tyr Val Arg Pro Val SerSer Trp Lys Val Ala Val Phe Glu His Gln 1 5 10 15 Val Ile Pro Pro LysThr Asp Met Glu Thr Arg Glu Glu Ala Leu Asp 20 25 30 Ala Leu Lys Leu AsnSer Asp Val Tyr His Glu Ala Val Leu Glu Ser 35 40 45 Arg Ser Lys Gly ValLys Met Ile Val Phe Pro Glu Tyr Gly Leu Tyr 50 55 60 Asp Ile Asn Thr LeuThr Arg Thr Arg Met Asp Leu Met Ala Glu Lys 65 70 75 80 Val Pro His ProLys His Gly His Arg Asn Pro Cys Asp Glu Pro Glu 85 90 95 Tyr Gln Thr GlnSer Ser Glu Met Leu Arg Thr Phe Ser Cys Met Ala 100 105 110 Lys Glu AsnAsp Met Tyr Met Val Val Asn Met Ala Gly Arg Glu Pro 115 120 125 Cys ArgArg Ala Thr Glu Pro Glu Cys Pro Gly Asp Lys Gln Leu Leu 130 135 140 TyrAsn Thr Asn Val Ala Phe Asn Asn Glu Gly Asp Val Val Ala Arg 145 150 155160 Tyr Tyr Lys Thr His Leu Phe Trp Glu Glu Gly Trp Phe Asn Ser Ser 165170 175 Lys Asn Tyr Glu Met Ala Leu Trp Asp Thr Pro Ile Gly Lys Phe Gly180 185 190 Thr Phe Met Cys Phe Asp Phe Gln Ala Val Gln Leu Ile Glu GlnTyr 195 200 205 Asn Val Arg His Ile Ala Tyr Pro Ala Ser Trp Val Asn LeuPro Pro 210 215 220 Ile Tyr Gln Ser Ile Gln Ser His Ser Ala Phe Ala ArgPhe Ala Lys 225 230 235 240 Ile Asn Leu Leu Ala Ala Ser Val His Arg LeuGlu Thr Ser Thr Tyr 245 250 255 Gly Ser Gly Ile Tyr Ser Pro Asn Gly AlaGlu Ile Phe Tyr Phe Arg 260 265 270 Pro Asp Ile Pro Lys Ser Lys Leu LeuVal Ala Glu Ile Leu Pro Ile 275 280 285 His Val Lys Lys Pro Glu Gln ThrVal Val Asn Lys Asp Asn Pro Val 290 295 300 Phe Pro Ser Glu Asp Asp AspVal Gln Asp Leu Phe Asp Arg Gly Asp 305 310 315 320 Phe Ala Phe Leu LysTyr Lys Arg Met Thr Thr Arg Ala Gly Thr Val 325 330 335 Glu Val Cys GlnLys Ser Phe Cys Cys Lys Ala Arg Tyr Ala Val Lys 340 345 350 Asp Arg PheLys Glu Val Tyr Ala Val Gly Val Tyr Asp Gly Leu Leu 355 360 365 Ser AlaGly Ala Asn Asn Leu Tyr Phe Gln Ile Cys Thr Val Ile Gln 370 375 380 CysPro His Lys Lys Cys Gly Leu Lys Ile Ser Lys Val Arg Thr His 385 390 395400 Phe Lys Tyr Leu Asn Leu Arg Ala Asp Gly Trp Leu Asp Arg Tyr Val 405410 415 Phe Pro Ser Tyr Thr Val Met Tyr Asn Asn Tyr Ile Ala Leu Asp Pro420 425 430 Phe Val Trp Asn Tyr Thr Val Ala Gly Gly Ile Glu Thr Lys ProGly 435 440 445 Thr Ser Thr Pro Leu His Ser Ala Asn Leu Val Ala Arg IleTyr Ala 450 455 460 Lys Asp Ser Ser Lys His Val His Gln Pro His Pro IleAsp Glu Gly 465 470 475 480 Val Ile Lys Met Ala Val Lys Tyr Met Leu TyrVal Met Ala Ala Tyr 485 490 495 Val Tyr Ala Ala Ser 500 2 370 PRTSymplectoteuthis oualaniensis 2 Ala Thr Glu Pro Glu Cys Pro Gly Asp LysGln Leu Leu Tyr Asn Thr 1 5 10 15 Asn Val Ala Phe Asn Asn Glu Gly AspVal Val Ala Arg Tyr Tyr Lys 20 25 30 Thr His Leu Phe Trp Glu Glu Gly TrpPhe Asn Ser Ser Lys Asn Tyr 35 40 45 Glu Met Ala Leu Trp Asp Thr Pro IleGly Lys Phe Gly Thr Phe Met 50 55 60 Cys Phe Asp Phe Gln Ala Val Gln LeuIle Glu Gln Tyr Asn Val Arg 65 70 75 80 His Ile Ala Tyr Pro Ala Ser TrpVal Asn Leu Pro Pro Ile Tyr Gln 85 90 95 Ser Ile Gln Ser His Ser Ala PheAla Arg Phe Ala Lys Ile Asn Leu 100 105 110 Leu Ala Ala Ser Val His ArgLeu Glu Thr Ser Thr Tyr Gly Ser Gly 115 120 125 Ile Tyr Ser Pro Asn GlyAla Glu Ile Phe Tyr Phe Arg Pro Asp Ile 130 135 140 Pro Lys Ser Lys LeuLeu Val Ala Glu Ile Leu Pro Ile His Val Lys 145 150 155 160 Lys Pro GluGln Thr Val Val Asn Phe Asp Asn Pro Val Phe Pro Ser 165 170 175 Glu AspAsp Asp Val Gln Asp Leu Phe Asp Arg Gly Asp Phe Ala Phe 180 185 190 LeuLys Tyr Lys Arg Met Thr Thr Arg Ala Gly Thr Val Glu Val Cys 195 200 205Gln Lys Ser Phe Cys Cys Lys Ala Arg Tyr Ala Val Lys Asp Arg Phe 210 215220 Lys Glu Val Tyr Ala Val Gly Val Tyr Asp Gly Leu Leu Ser Ala Gly 225230 235 240 Ala Asn Asn Leu Tyr Phe Gln Ile Cys Thr Val Ile Gln Cys ProHis 245 250 255 Lys Lys Cys Gly Leu Lys Ile Ser Lys Val Arg Thr His PheLys Tyr 260 265 270 Leu Asn Leu Arg Ala Asp Gly Trp Leu Asp Arg Tyr ValPhe Pro Ser 275 280 285 Tyr Thr Val Met Tyr Asn Asn Tyr Ile Ala Leu AspPro Phe Val Trp 290 295 300 Asn Tyr Thr Val Ala Gly Gly Ile Glu Thr LysPro Gly Thr Ser Thr 305 310 315 320 Pro Leu His Ser Ala Asn Leu Val AlaArg Ile Tyr Ala Lys Asp Ser 325 330 335 Ser Lys His Val His Gln Pro HisPro Ile Asp Glu Gly Val Ile Lys 340 345 350 Met Ala Val Lys Tyr Met LeuTyr Val Met Ala Ala Tyr Val Tyr Ala 355 360 365 Ala Ser 370 3 1646 DNASymplectoteuthis oualaniensis 3 ttcgagcatc aggtcattcc tccaaaaacagatatggaga ctcgagagga ggccctggac 60 gcactcaaat tgaatagcga tgtctatcatgaagcagtgc ttgaatcaag atcaaaagga 120 gtcaaaatga ttgtcttccc cgaatatggtctttatgata tcaacacatt gacaagaacg 180 aggatggacc tgatggcaga gaaagttccacaccctaaac acggccaccg aaacccatgc 240 gatgagccag agtatcaaac tcaaagttcagagatgttga gaactttcag ttgtatggcg 300 aaagaaaatg atatgtatat ggtggtcaacatggccggtc gtgagccatg taggcgtgcc 360 actgaacctg agtgcccagg agacaagcaattgctataca acactaatgt tgcttttaac 420 aatgaaggtg acgttgttgc aaggtattacaaaacccatc tattttggga agaaggttgg 480 ttcaactcgt ccaagaatta cgaaatggcactttgggaca ccccgattgg caaatttggt 540 acttttatgt gttttgactt ccaggctgttcaactaatcg aacaatataa cgtacgacat 600 attgcttacc cagcttcctg ggttaatctgcccccaatct atcaatcaat ccaatcccat 660 tcagcttttg ctcgttttgc aaagattaatctattggcag caagtgtcca caggctagag 720 accagtacct atggtagcgg catatattcacctaatggag ctgaaatctt ttatttccgt 780 ccagacattc caaaaagtaa actgttagtggccgagattt taccaatcca tgttaaaaaa 840 ccagagcaga cggtagtcaa ttttgataatcccgtattcc cgtctgaaga tgatgacgtg 900 caagatcttt ttgatcgggg cgactttgctttcttaaaat ataaacgtat gacaaccagg 960 gctggtacag tggaagtttg ccaaaagtccttctgttgca aagctcgcta tgcagtcaaa 1020 gatcgattca aagaagttta cgctgtgggtgtctatgacg gtttattatc tgccggtgcc 1080 aacaatctct acttccagat ttgtacggtgattcaatgtc ctcataagaa gtgcggactg 1140 aaaatatcaa aagtaaggac tcatttcaagtaccttaatt tgcgagcaga tggatggtta 1200 gatagatacg tgtttccttc atatacggtaatgtacaaca attacattgc gcttgatcca 1260 tttgtatgga attatacagt ggccggaggaatcgaaacta aaccaggcac aagcactcct 1320 ctacattcag ccaatctggt cgcaaggatttatgcaaaag acagttcaaa gcacgtccat 1380 cagccacatc ctattgacga aggcgtaattaaaatggccg ttaaatatat gttatatgta 1440 atggcagctt atgtttacgc cgcctcctaaatatcgaaaa taaacattgg accactcgaa 1500 aacctcatcg aatgatgatg ctgaaagattagggatttat gaataatgct ttttcaattc 1560 ttggtggtgg tgctgcttaa aaaattatgcgaatactttg cacgaacttt aaaagtttag 1620 atcaagatga tttccatgaa aaaatc 16464 28 DNA Artificial Sequence Primer for PCR cloning 4 cgggatccttygarcaycar gtnathcc 28 5 28 DNA Artificial Sequence Primer for PCRcloning 5 gcctcgagtc rtrrttraan gcnacrtt 28 6 20 DNA Artificial SequencePrimer for PCR cloning 6 atggacctga tggcagagaa 20 7 26 DNA ArtificialSequence Primer for PCR cloning 7 tgrtadatng gnggnargtt naccca 26 8 20DNA Artificial Sequence Primer for PCR cloning 8 gtytgnacrt crtcrtcytc20 9 23 DNA Artificial Sequence Primer for PCR cloning 9 gagaccagtacctatggtag cgg 23 10 28 DNA Artificial Sequence Primer for PCR cloning10 ttgaattccc aaaaacagat atggagac 28 11 28 DNA Artificial SequencePrimer for PCR cloning 11 ttgaattcct attttgggaa gaaggttg 28 12 31 DNAArtificial Sequence Primer for PCR cloning 12 aagtcgactt aggaggcggcgtaaacataa g 31

1. A protein derived from Symplectoteuthis oualaniensis (okinawan squid)having the following characteristics: (1) emitting luminescence atwavelength of 470 nm by binding to dehydrocoeleneterazine or adehydrocoeleneterazine derivative in the presence of potassium ion orsodium ion: (2) having a molecular mass of approximately 60 kDa analyzedby SDS-polyacrylamide gel electrophoresis: (3) dissolving into a solventcontaining KCl at a concentration of 0.6 M or higher while maintainingability to emit luminescence: and (4) decomposing by trypsin digestionto produce fragments having molecular masses of 40kDa and 15kDa.
 2. Theprotein according to claim 1, wherein said dehydrocoeleneterazinderivative is a compound selected from the group consisting of compoundsrepresented by the following formula (1), (2) or (3):

wherein X is a halogen atom or methoxy group, a compound represented bythe following formula:

wherein X represents a halogen atom, methoxy group or hydroxyl group,and Y and Z each represents hydrogen atom, a halogen atom, methoxy groupor hydroxyl group, with the proviso that a case where both of Y and Zare hydrogen atoms is excluded, and a compound represented by thefollowing formula:

wherein X and A each represents a halogen atom, methoxy group orhydroxyl group, and Y, Z, B and C each represents hydrogen atom, ahalogen atom, methoxy group or hydroxyl group, with the proviso thatcases where Y and Z are substituents selected from an atom or groupsdescribed above with both of B and C being hydrogen atoms and the casewhere all of Y, Z, B and C are hydrogen atoms are excluded.
 3. A proteinderived from Symplectoteuthis oualaniensis (okinawan squid) consistingof an amino acid sequence of following (a) or (b): (a) an amino acidsequence represented by amino acid numbers 1 to 501 shown in SEQ:ID NO:1in a sequence list, or (b) an amino acid sequence in which a part ofsaid amino acid sequence (a) is deleted or another amino acid sequenceis added to said amino acid sequence (a) or a part of amino acidsequence (a) is substituted with another amino acid sequence, the aminoacid sequence (b) having characteristics that binds withdehydrocoeleneterazine by covalent bonding in the presence of potassiumion or sodium ion thereby emitting luminescence.
 4. A protein derivedfrom Symplectoteuthis oualaniensis (okinawan squid) consisting of anamino acid sequence of following (c) or (d): (c) an amino acid sequencerepresented by amino acid numbers 1 to 370 shown in SEQ:ID NO:2 in asequence list, or (d) an amino acid sequence in which a part of saidamino acid sequence (c) is deleted or another amino acid sequence isadded to said amino acid sequence (c) or a part of amino acid sequence(c) is substituted with another amino acid sequence, the amino acidsequence (d) having characteristics that binds withdehydrocoeleneterazine by covalent bonding in the presence of potassiumion or sodium ion thereby emitting luminescence.
 5. A method fordetection of a monovalent cation, the method comprising formation of aconjugate adduct comprising the protein according to claim 3 or 4 and anchromophore, thereby emitting luminescence from said conjugate adduct inthe presence of a monovalent cation.
 6. The method according to claim 5,wherein said chromophore is a compound selected from the groupconsisting of compounds represented by the following formula (4), (5),(6) or (7):

a compound represented by the following formula:

wherein X is a halogen atom or methoxy group, a compound represented bythe following formula:

wherein X represents a halogen atom, methoxy group or hydroxyl group,and Y and Z each represents hydrogen atom, a halogen atom, methoxy groupor hydroxyl group, with the proviso that a case where both of Y and Zare hydrogen atoms is excluded, and a compound represented by thefollowing formula:

wherein X and A each represents a halogen atom, methoxy group orhydroxyl group, and Y, Z, B and C each represents hydrogen atom, ahalogen atom, methoxy group or hydroxyl group, with the proviso thatcases where Y and Z are substituents selected from an atom or groupsdescribed above with both of B and C being hydrogen atoms and the casewhere all of Y, Z, B and C are hydrogen atoms are excluded.
 7. A geneencoding the protein according to claim 3 or
 4. 8. A gene derived fromSymplectoteuthis oualaniensis (okinawan squid) consisting of a basesequence of following (e), (f) or (g): (e) a base sequence representedby base numbers 1 to 1646 shown in SEQ:ID NO:3 in a sequence list, (f) abase sequence in which a part of said base sequence (e) is deleted oranother base sequence is added to said base sequence (e) or a part ofbase sequence (e) is substituted with another base sequence, the basesequence (f) encoding a protein having characteristics that binds withdehydrocoeleneterazine by covalent bonding in the presence of potassiumion or sodium ion thereby emitting luminescence, or (g) a base sequencethat hybridizes with the base sequence (e) under stringent conditions.9. A method for detection of a monovalent cation, the method comprisingintroduction of the gene according to claim 7 or 8 into a cell,expression of protein encoded by said gene in the cell, formation of aconjugate adduct of the protein and a chromophore, thereby emittingluminescence from said conjugate adduct in the presence of a monovalentcation.
 10. The method according to claim 9, wherein said chromophore isa compound selected from the group consisting of compounds representedby the following formula (8), (9), (10) or (11):

a compound represented by the following formula:

wherein X is a halogen atom or methoxy group, a compound represented bythe following formula:

wherein X represents a halogen atom, methoxy group or hydroxyl group,and Y and Z each represents hydrogen atom, a halogen atom, methoxy groupor hydroxyl group, with the proviso that a case where both of Y and Zare hydrogen atoms is excluded, and a compound represented by thefollowing formula:

wherein X and A each represents a halogen atom, methoxy group orhydroxyl group, and Y, Z, B and C each represents hydrogen atom, ahalogen atom, methoxy group or hydroxyl group, with the proviso thatcases where Y and Z are substituents selected from an atom or groupsdescribed above with both of B and C being hydrogen atoms and the casewhere all of Y, Z, B and C are hydrogen atoms are excluded.
 11. Atransformed Escherichia coli wherein the gene according to claim 7 orclaim 8 is introduced.
 12. A method to produce a recombinant protein,the method comprising introduction of the gene according to claim 7 orclaim 8 succeeded by expression of protein encoded by said gene inEscherichia coli, said recombinant protein binds withdehydrocoeleneterazine by covalent bonding in the presence of potassiumion or sodium ion to form a conjugate adduct, thereby emittingluminescence from said conjugate adduct.